JP2001513367A - Temperature-controlled pH-dependent formation of ionic polysaccharide gels - Google Patents

Abstract

The present invention relates a temperature-controlled pH-dependant formation of ionic polysaccharide gels, such as chitosan/organo-phosphate aqueous systems, and methods of preparation thereof. While chitosan aqueous solutions are pH-dependant gelating systems, the addition of a mono-phosphate dibasic salt of polyol or sugar to a chitosan aqueous solutions leads to further temperature-controlled pH-dependant gelation. Solid organo-phosphate salts (1-20% w/v) are added and dissolved at low temperature (10° C.) within 0.5 to 4.0% w/v chitosan in aqueous acidic solutions. Aqueous chitosan/organo-phosphate solutions are initially stored at low temperatures (4° C.), then endothermally gelated within the temperature range of 30 to 60° C. Chitosan/organo-phosphate solutions rapidly turn into gels at the desired gelation temperature. Gelation can be ex vivo within any receivers or molds, or in situ in animals or humans (in vivo) so as to fill a tissue defect or cavity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] BACKGROUND OF THE INVENTION (a) Field of the Invention The present invention, ionic polysaccharide gels such as chitosan / organic - Temperature control pH dependent forming phosphate saltwater systems, and methods for their preparation. (B) Description of the Prior Art Chitosan is a commercially available inexpensive polymer, a derivative of chitin or poly (N-acetyl-glucosamine) material. Chitosan is a marine animal (fish, crustaceans, shrimp,
Mainly derived from the D-glucosamine units extracted from the hard shell of crabs, etc., or generated through the catalyzed N-deacetylation of chitin, an insoluble biopolymer synthesized from natural organisms (zygote, mold, etc.). Become. Chitosan is expected to have good viscoelasticity and has sufficient histocompatibility and biodegradability to make it ideal for bioactive and resorbable implants. Poly-D-glucosamine chains have the ability to attach to numerous proteoglycan molecules and are also known to coexist with fibrous collagen forming aqueous gels. It is believed that the role of the proteoglycans in the gel is to retain water and provide proper viscoelasticity. The resulting extracellular matrix is expected to provide a compatible environment for cell proliferation and tissue formation, especially skin, ligament, bone and chondrocytes. As a result, chitosan has attracted great interest as a scaffold or support material for bioengineered artificial tissues.

[0002] In addition, chitin and partially acetylated chitosan have been extensively investigated for therapeutic or implantable materials. The biocompatibility of chitosan-based materials has been studied, especially for blood, wounds and bone. The immunological and genotoxic activity as well as the stimulatory effect on macrophage action have also been studied using various chitosan substances.

[0003] Chitosan and its derivatives have been explored for drug delivery systems through gels (Ohya Y. et al. (1993) J. Micro-encapsulation, 10 (1): 1-9). Delivery of peptides using chitosan has been proposed to act intranasally. Cationic colloid drug carriers have been proposed from the chitosan-polycaprolactone system. Wound healing and reconstruction devices made with chitosan materials have been proposed for open or corneal wounds, periodontal tissue and skin. Chitosan has been specifically studied in bone and dura matter and as a hemostatic agent.

[0004] The entrapment of living biologicals (cells, enzymes, etc.) has been investigated using separate chitosan products; however, in almost all cells, living organisms have alginate / chitosan microbeads. It has been sealed inside. The inclusion of cartilage cells has been proposed in calcium-alginate / chitosan beads.

[0005] Gelation of chitosan through polyphosphate has been encouraged to encapsulate cells such as nerve or muscle squeletal tissue. In general, chitosan in an acid / aqueous medium was loaded with the cell suspension and the resulting mixture was dropped into buffered pentasodium tripolyphosphate to form cell-loaded chitosan beads and capsules. Uptake of neurons in polyphosphate-gelled chitosan beads led to good cell viability, but to a low growth rate (
Zielinski B. A. et al. (1994) Biomaterials, 15 (13): 1049-1056). Large or specific three-dimensional forms of the substance were not proposed (Zielinski B. A. et.
al. (1994) Biomaterials, 15 (13): 1049-1056). Polysaccharide capsules have been proposed to take up physiologically active cells such as islets of Langerhans (US Pat. No. 4,391,909). Chitosan / cisplatin hydrogen chloride
) The mixture was cross-linked and proposed as a drug delivery system.

[0006] Chitosan derivatives have been incorporated into many carrier compositions or drug formulations. Chitosan materials such as wound filling materials or contraceptive products have also been proposed (US Pat. No. 4,956,35).
0 and 4,474,769). Chitosan gel was once again proposed as a support for immobilizing and encapsulating living biomaterials, such as cells, bacteria and mold (US Pat. No. 4,647,536). An ocular drug delivery system made by chitosan has also been proposed for in situ gelling and molding (US Pat. No. 5,422,116).

In US Pat. No. 4,659,700, chitosan gel was produced from a glycerol / acid / water system as a biodegradable carrier for drug delivery. The resulting chitosan gel was reported to be quite stable over a long period of time and at a wide range of temperatures, especially at 4 to 40 ° C., retaining a perfect three-dimensional morphology. Gels and gel-like materials were processed by dissolving 1.1 to 4.0% w / v chitosan in an acid-water-glycerol solution, wherein acetic acid, formic acid or propionic acid and 10-90 It is preferred to use a% glycerol ratio and neutralize with a liquid base such as sodium, ammonium and potassium hydroxide or ammonia vapor. The pH of the resulting chitosan-glycerol gel material was about pH 7.0.
It is. After neutralization, the resulting material is turned into a gel upon standing, and such gels are apparently caused by the interaction of chitosan, glycerol and water. Free glycerol or water was apparently not reported. Notably, however, such a three-dimensional form of chitosan-glycerol gel only occurs if the solution has been previously neutralized with a base. A piece of three-dimensional morphological gel can be easily laminated, as well as be a gel-like membrane. The role of the glycerol component and the chitosan-glycerol interaction has not been determined.

[0008] Gels formed in situ have been proposed in US Pat. No. 5,587,175 with ionic polysaccharides. The composition can be used as a medical device for drug delivery, application of diagnostic agents, or prevention of post-operative adhesion, and consists of an aqueous liquid medium that can gel in situ. It comprises at least one ionic polysaccharide, at least one film-forming polymer, and a drug or pharmaceutical agent, water, and optionally, counter ions capable of gelling the ionic polysaccharide (US Pat. No. 5,587,175). . However, gelation is achieved by interaction between the ionic polysaccharide and the film-forming polymer, or by counterion-induced cross-linking of the ionic polysaccharide. Another in situ molded gel is a polyoxyalkylene composition (US Pat. No. 4,185,618) or a polyoxyalkylene / polysaccharide mixture (US Pat. No. 5,126,141) or an alginic acid / cationic mixture in situ (US Pat. Nos. 4,185,618 and 5,266,326).

It is highly desirable to provide a temperature-controlled, pH-dependent shaped polysaccharide gel that can be used to encapsulate cells and cellular material while retaining their biological activity.

It is highly desirable to provide such a polysaccharide gel that retains its solid state or gel state at physiological temperature or 37 ° C. SUMMARY OF THE INVENTION One object of the present invention is to provide a temperature-controlled, pH-dependent, form that can be used to encapsulate cells and cellular material while retaining their biological activity.
ed) to provide a polysaccharide gel.

[0011] Another object of the present invention is to provide such a polysaccharide gel that retains its solid state or gel state at physiological temperature or 37 ° C. Another object of the present invention is to provide a method for producing such a polysaccharide gel.

According to the present invention, a) 0.1 to 0.5% by weight of chitosan or a chitosan derivative; and b) 1.0 to 2.0% by weight of a dibasic monophosphate of a polyol or a sugar. A gel based on a polysaccharide comprising a salt of a polyol or a sugar selected from the group consisting of monosulfates and monocarboxylates, provided that said gel is derived and stable in the range of 20 to 70 ° C. And adapted to be formed and / or gelled in situ within animal or human tissues, organs or cavities.

The salt may be any of the following or any combination of the following: a) glycerol-2-phosphate, sn-glycerol 3-phosphate and L-
Dibasic monophosphate selected from the group consisting of glycerol including salts of glycerol-3-phosphate; b) dibasic monophosphate and the polyol are histidine, acetol, diethylstilbestrol, indole-glycerol. Sorbitol, ribitol, xylitol, arabinitol, erythritol, inositol, mannitol, glucitol and mixtures thereof; c) dibasic monophosphate and the sugar are fructose, galactose, ribose, glucose, xylose, Rhamnulose, sorbose, erythrulose, deoxy-ribose, ketose, mannose, arabinose, fucrose, fructopyranose, ketoglucose, sedoheptulose, trehalose, tagatose, sucrose , Allose, threose, xylulose, hexose, methylthio-ribose, methylthio-deoxy-ribulose and mixtures thereof; d) dibasic monophosphate and the polyol are palmitoyl-glycerol, linoleoyl-glycerol, Oleoyl-glycerol, arachidonoyl-
And e) the glycerophosphate is selected from the group consisting of disodium glycerophosphate, dipotassium glycerophosphate, calcium glycerophosphate, barium glycerophosphate and strontium glycerophosphate.

[0014] Preferred gels according to one embodiment of the present invention are chitosan-β-glycerophosphate, chitosan-α-glycerophosphate, chitosan-glucose-1-glycerophosphate, and chitosan-fructose-6-glycerophosphate. Selected from the group consisting of:

[0015] Solid particulates or water-soluble adducts may be incorporated with the polysaccharide gel before gelation. Drugs, polypeptides or non-viable pharmaceutical agents may be incorporated with the polysaccharide gel prior to gelation.

[0016] Surviving microorganisms, plant cells, animal cells or human cells may be encapsulated with the polysaccharide gel before gelation. The gel may be formed in situ, subcutaneously, intraperitoneally, intramuscularly or in a connective tissue of an organism, bone defect, fracture, joint cavity, body conduit or cavity, ocular cul-de-sac, or solid tumor.

The gels of the present invention may be used as carriers for delivering pharmaceutical agents in situ. According to the present invention there is also provided a method for producing a polysaccharide gel solution according to the present invention, comprising the following steps: a) Aqueous polysaccharide having a concentration of 0.1 to 5.5% by weight of chitosan or chitosan derivative. Dissolving the chitosan or chitosan derivative in an aqueous acidic solution at a pH from about 2.0 to about 5.0 to obtain a composition; and b) a salt of a polyol or a sugar, the dibasic monophosphate salt From 1.0 to 20% by weight of a salt selected from the group consisting of monobasic dibasic salt and monocarboxylate
Dissolving in the aqueous polyol composition of step a) a polysaccharide gel solution is obtained, wherein the polysaccharide gel has a concentration of 0.1 to 5.0% by weight of chitosan or a chitosan derivative and 1.0% by weight. Having a concentration of from about 20 to about 20% by weight of a polyol or sugar and having a pH of from about 6.4 to about 7.4.

The method includes, after step b), step c): c) heating the polysaccharide gel solution at a solidification temperature ranging from about 20 ° C. to about 80 ° C. until formation of the polysaccharide gel. Is fine.

A pharmaceutical agent may be added to the polysaccharide gel solution of step b). The method comprises, after step b), step i): i) distributing the polysaccharide gel solution in a mold or in any of the tissues, organs or body cavities to a desired receptor for gelation. May be further included.

The aqueous acidic solution includes acetic acid, ascorbic acid, salicylic acid, phosphoric acid, hydrochloric acid,
It may be prepared from an organic or inorganic acid selected from the group consisting of propionic acid, formic acid, and mixtures thereof.

[0021] The polysaccharide gel solution may be maintained in a stable non-gelling liquid form at a temperature ranging from about 0 ° C to about 20 ° C. Solidification temperatures range from about 37 ° C to about 60 ° C, preferably about 37 ° C.

[0022] The molecular weight of chitosan ranges from about 10,000 to 2,000,000.
The polysaccharide gel can be heated by adjusting the pH of the polysaccharide gel when the pH of the polysaccharide gel solution is> 6.9 or when the pH of the polysaccharide gel solution is <6.9. It becomes irreversible or temperature reversible.

A solid particulate adduct may be added to the polysaccharide gel solution of step b). The polysaccharide gel solution may be introduced into an animal or human body by injection or endoscopic administration, or may gel in situ at a temperature of about 37 ° C.

According to the present invention, there is also provided the use of a polysaccharide gel to produce a biocompatible degradable substance for use in cosmetics, pharmacology, drugs and / or surgery.

The gel, as a whole or as a component, may be incorporated into a device or implant that can be implanted in either an animal or human for the repair, reconstitution and / or replacement of tissues and / or organs.

The gel may be used as an implantable, transdermal, or dermal drug delivery system as a whole or as an ingredient. The gel may be used as a whole or as an ingredient in an ophthalmic implant or drug delivery system.

The gel may be used to produce a cell-loaded artificial matrix applied to the engineering and culture of biotechnological hybrid materials and tissue equivalents. The loaded cells are chondrocytes (indirect chondrocytes), fibrochondrocytes (meniscus), ligament fibroblasts (ligaments), skin fibroblasts (skin), tendon cells (tenocytes)
(Tendon), myofibroblasts (muscle), mesenchymal stem cells and keratinocytes (skin).

The cell-loaded gels and derived products are dedicated to the culture or engineering of artificial joint chondrocytes and chondrocyte-like tissues and organs, either for surgical or laboratory testing applications. .

Cell-loaded gels and derived products are used in the processing or engineering of living artificial substitutes for ligaments, tendons, skin, bone muscle and any metabolic organs, surgical or laboratory testing applications Dedicated for any of the

[0030] The cell-loaded gel and the derived product are used for articular chondrocytes, fibrochondrocytes,
It is applied as a living alternative for the replacement of chondrocyte-like organs, ligaments, tendons, bone tissue or skin.

The cell-loaded hydrogel induces ectopic formation of fibrochondrocyte-like or chondrocyte-like tissue by being gelled in situ. According to the present invention, an injectable that acts as a support, carrier, reconstitution device or alternative for in situ shaping of bone-like, fibrochondrocyte-like or chondrocyte-like tissue in animal or human physiological locations Alternatively, an implantable gel-like biomaterial is also provided.

The polysaccharide gel solution comprises: 1) incorporating and dissolving at least one complementary polymer in the polysaccharide gel solution; and 2) 20 ° C. to 60 ° C.
May be used to produce a derivatized gel or hydrogel by allowing the polysaccharide gel solution to interact with the complementary polymer for a time sufficient to form a transparent three-dimensional gel within a temperature range of

The complementary gel is a non-ionic water-soluble polysaccharide or hydroxylalkyl cellulose. For the purposes of the present invention, the following terms and expressions are defined as follows.

The term “polysaccharide gel solution” refers to a non-gel that is stable at a temperature in the range of about 0 ° C. to about 15 ° C. that can gel or change to a gel state when heated at the gelling temperature. It is intended to mean a polysaccharide solution in a fluorinated liquid form.

The term “gelling temperature” is intended to mean any temperature ranging from about 20 ° C. to about 80 ° C., preferably from about 37 ° C. to about 60 ° C., and more preferably about physiological temperature Or 37 ° C.

The expression “polyol or sugar salt” is intended to mean monobasic, monobasic and monocarboxylic acid salts of polyols or sugars. The present invention includes a method of forming separate gelled materials, wherein the materials are shaped (customized forms, tubes, membranes, films, etc.) or formed in situ in a biological environment (Filling of tissue defects).

According to a preferred embodiment, the chitosan / organophosphate aqueous solution comprises chitosan pK
It has a pH greater than a and changes to a solid gel upon temperature stimulation. The polyol gel can be used as a drug carrier or as a non-viable therapeutic delivery system, as a tissue and organ replacement, and as an inclusion body for living cells or microorganisms. The chitosan / organo-phosphate gel matrix is 30
Molded at a temperature of between 60 and 60 ° C. The chitosan / organo-phosphate aqueous system is used as an injectable filling material and is injected and filled in situ to fill and repair tissue defects.

Glycerol-2-phosphate, glycerol-3-phosphate and glucose-1
-Salts based on phosphoric acid are the preferred salts disclosed according to the invention. Chitosan / polyol- or sugar-phosphate and chitosan / polyol- or sugar-sulfate gels can be applied for surgical reconstitution and regeneration applications as well as for drug delivery purposes. They provide thermoreversible or irreversible bioerodible polymeric gels with well-known biologically compatible components for a wide range of medical / biotechnological applications. DETAILED DESCRIPTION OF THE INVENTION By dissolving chitosan in an acidic aqueous solution, a p in the range of 4.3 to 5.6 is obtained.
A clear aqueous chitosan solution having H levels is obtained. The chitosan solution is sterilized by filtration or steam autoclave and stored at low plus temperature (4 ° C.). The organic-phosphate component is added to the chitosan solution, preferably at a low positive temperature (4 ° C.), and then the aqueous chitosan / organo-phosphate mixture is heated at a temperature in the range of 30 to 60 ° C. through an endothermic mechanism. Gel. Once formed, the resulting chitosan / organo-phosphate gel is thermally stable even to heating up to 180 ° C (in an autoclave), especially in cell culture media. Bioencapsulation of the chitosan / organo-phosphate gel is obtained by incorporating viable cells into a non-gelled aqueous chitosan / organo-phosphate solution at low temperature (4 ° C). Next, if the temperature of the chitosan / organo-phosphate / cells of the resulting mixture is raised and maintained at 37 ° C., gelation occurs within one hour. Organic-sulfates or monocarboxylates of polyols or sugars have a similar role to organic-phosphates.

[0039] Chitosan and its derivatives are relatively inexpensive commercially available materials and represent an attractive group of biocompatible and degradable polymers. They have solid or solution properties that can be modified by changing their chemical composition and / or physico-chemical properties. The degree and molecular weight of deacetylation has been shown to greatly affect solution properties, enzymatic degradation and biological activity. Chemical modifications have been proposed to neutralize or modify the chitosan chain, for example, by incorporating carboxylic acid, acetate, glutamic acid, carboxymethyl or sulfate groups. Chemical cross-linking of macromolecules of chitosan (anhydride, glutaraldehyde, succinimide glutamate-PEG, etc.) creates a network of branching or grafting by inducing covalent bonds.

Physical gelation of chitosan and its derivatives is another technique: a) neutralization (NaOH, KOH, NH ₄ OH ...) that induces hydrogen bonds between the chitosan chains; b) pure static Ionic complications using divalent anions (borate, molybdate, polyphosphate, sulfate and sulfated macromolecules ...) to induce electrical interactions; c) electrostatic interactions and interfaces It can be obtained by using complications with anionic surfactants (sodium alkyl sulphate ...) which induce a surfactant-surfactant hydrophobic interaction.

According to the present invention, a novel gelling mechanism is proposed that combines hydrogen bonding, electrostatic interaction and chitosan-chitosan hydrophobic interaction. It is a chitosan molecule,
It can only be achieved through a complex interaction between the water molecule and the dibasic monophosphate of the polyol or sugar.

[0042] Polyols are frequently added to the composition to improve gel properties. Sorbitol and mannitol are currently used as tonicity enhancing agents. Glycerol and polyethylene glycol are proposed as plasticizers. Polyols (-ol: glycerol, sorbitol ...) and sugars (-ose: fructose, glucose, galactose ...) were used as temperature stabilizers for proteins in solution (Back JF et al. (1979) Biochemistry, 18 (23): 5191
−5196). Depending on the molecule selected, they have been found to create or break the structure of water, create hydrogen bonds, interact electrostatically or hydrophobically, and exhibit endothermic transitions ( Back JF et al. (1979) Biochemistry, 18 (23):
5191-5196). Polyols and sugars stabilize proteins against thermal denaturation by enhancing their structural effects on water as well as hydrophobic interactions.

Beta-glycerophosphate disodium or calcium salt, or glycerol-2-phosphate disodium or calcium salt, is a well studied molecule in biological science. It is considered a substrate for alkaline phosphatase (AL). Glycerophosphate is widely used as a cell culture media adjunct for culturing cells isolated from musculoskeletal tissue and, when delivered to bone / chondrocytes during culturing, synthesizes certain matrix components. (Chung C.-H. et al. (1992) Calcif. Tissue Int., 51: 305-311; Bellows C. G. et al. (1992) Bone). and Mineral, 17: 15-29). Although gelation of chitosan occurs with glycerophosphate of any grade or purity, encapsulation of living biologicals requires glycerophosphate tested in cell culture. Disodium or calcium salts of alpha-glycerophosphate or disodium or calcium glycerol-3-phosphate are also biologically important organic salts (Chung C.-H. et al. (1992) Calcif. Tissue Int. , 51: 305-3
11). Glycerophosphate is obtained from glycerophosphate obtained through hydrolysis of lecithin, a phosphatide that is a well-known biomolecule of eggs, soy and fish. Glycerophosphate exists as two isomeric structures, alpha and beta, beta-glycerophosphate is optically inactive and alpha-glycerophosphate is optically active. Glycerophosphate is a physiologically active compound and is involved in carbohydrate metabolism. Glycerophosphate dehydrogenase was also found to be active in neural tissue, but glycerophosphate was reported to accelerate the rate of methylene blue detachment by guinea pig nerves. Alpha-glycerophosphate interacts with pyruvate through an oxidation-reduction reaction to produce lactic acid in fresh muscle extracts. Glycerophosphate is currently available in disodium, calcium,
It is commercially available as magnesium, dipotassium, strontium and barium salts with relatively strong basic properties. Both alpha and beta-glycerophosphates are inexpensive, readily available sources of organic monophosphate dibasic salts between polyols or sugar phosphates.

[0044] Solubilization of chitosan in aqueous solutions requires protonation of the amine groups of the chitosan chain to reach into acidic aqueous solutions having a pH in the range of 3.0 to 5.0. When solubilized, chitosan remains soluble until the pH is about 6.2. Neutralization of the acidic chitosan solution with alkali results in an increase in pH as well as deprotonation of the amine groups. Neutralization of the chitosan solution to a pH above the pKa of chitosan at about 6.3-6.4 results in OH-HN and O-HN inner chains, triggering a hydrated three-dimensional network, chitosan gel. . PH above 6.3-6.4
In, the chitosan solution systematically yields a chitosan gel in the normal temperature range (0-60 ° C.). However, the addition of the organophosphate to the aqueous chitosan solution increases the pH of the chitosan / organophosphate solution and remains liquid without being gelled for prolonged periods, even above 6.5 to 7.2. is there. This new-neutral chitosan / organophosphate aqueous solution (pH 6.5-7.2) becomes a gel when stimulated by sufficient temperature. Gelation time is controlled by temperature. For example, a chitosan / organophosphate solution that gels in about 30 minutes at 37 ° C. requires only 2 minutes at 60 ° C. to form a gel.

The mechanism of gelation as well as the properties of the gel are expected to be similar for all chitosan / organo-phosphate systems. Thus, the gelation of a chitosan / β-glycerophosphate solution, which has been investigated in more detail, can be considered to be a typical example. The results showing the pH and temperature dependence of gelation for the chitosan / β-glycerophosphate solution are summarized in the sol-gel diagrams shown in FIGS. In addition to gel strength, the flow experiments shown in FIG. 3 clearly show that upon heating, gelation of the chitosan / β-glycerophosphate solution occurs. The rate change that appears at about 60 ° C. is a precursor to the sol-to-gel transition and gel formation.
This temperature for gel formation will depend on the properties of the solution and the heating rate (the required activation energy).

The pore structure of the chitosan / β-glycerophosphate gel was verified by scanning electron microscopy, as shown in FIG. The gel contains about 100 microns of chamber and about 1
It has a typical microstructure with 0 micron pores. Their microstructure is
This differs from the structure observed in chitosan gels processed by simple neutralization when a lamellar conformation is present.

Other important properties relate to the injectability of the solution and the in vivo gelation of chitosan / β-glycerophosphate. Injections and gels are typically shown in FIGS. 5A and 5B. The black arrow in FIG. 5A indicates the gel formed in situ in the zone indirectly in the knee between the collateral ligament and the lower leg (patellar tendon). Other gels are formed subcutaneously between the skin and leg muscles. The black arrow in FIG. 5B indicates a gel formed in situ subcutaneously in the torso region.

In the chitosan / organo-phosphate gel, the organic-phosphate anion contributes to the cross-linking of the chitosan macromolecular chains, but is an inorganic divalent anion such as sulfate, oxalate, phosphate or It differs from the mode of pure ionic cross-linking that occurs during gelation of chitosan by polyphosphates (pyrophosphate, metaphosphate or tripolyphosphate). The chitosan aqueous solution immediately turns into a gel in the presence of the inorganic divalent anion and independent of the pH value of the solution. In addition, elevated temperatures constitute an unfavorable factor for the gelation of this type of system. In contrast, the gelation of the chitosan / organo-phosphate solution depends on both the final pH and the temperature of the chitosan / organo-phosphate solution. Any solution of chitosan / organo-phosphate has a pH
Can not gel at any temperature as long as is less than 6.45, and 6.4
Any solution of chitosan / organo-phosphate having a pH above 5 can be produced at 20 ° C without immediate gelling and at 4 ° C without changing to a gel
Can be stored for a long time. At 37 ° C., only chitosan / organo-phosphate solutions with a pH above 6.9 can gel somewhat more quickly. It is expected that the presence of organic-phosphate molecules in the chitosan solution will directly affect the electrostatic, hydrophobic, and hydrogen bonding of the chitosan chains. That is, the main interactions involved in the formation of chitosan / organo-phosphate gels are essentially: 1) chitosan / chitosan internal chain hydrogen bond (CHITOSAN-CHITOSAN); 2) ammonium groups on macromolecular chains and organic -Chitosan / organic-phosphate electrostatic attraction between the phosphate groups of the phosphate molecule (CHITOSAN
-PHOSPHATE); 3) resulting in a chitosan-chitosan hydrophobic interaction induced through the structural action of the polyol or sugar moiety on the water molecule. The structuring of the polyol portion of the water reduces the chitosan-water interaction and thus enhances the chitosan-chitosan interaction. An important aspect of such gelling derives essentially from the subsequent polyol-water-induced chitosan hydrophobic attraction that is enhanced upon increasing the temperature (temperature-controlled gelation). At low temperatures, strong chitosan-water interactions protect hydrated chitosan macromolecules from aggregation. Removal of water molecules during heating of the sheath
Assists and enhances chitosan-chitosan interactions, thus inducing macromolecular pairing. However, two first gravitational forces (CHITOSAN-CHITOSAN- and CHITOSAN-
If PHOSPHATE) is completely unusable in chitosan / organo-phosphate solutions, gelling will never occur. This explains the pH dependence that still governs the temperature controlled gelation of the chitosan / organo-phosphate system. Such CHITOSAN-PHOSPHATE
Although there is an electrostatic attraction, the phosphate group cannot be the only crosslinker of the chitosan chain due to the interference of incompatible stearic acid. This distinguishes this gelling mechanism from the purely ionic gelation of chitosan by phosphate or polyphosphate divalent anions. Pure ionic cross-linking should not be temperature-controlling or irritating.

This type of temperature-controlled pH-dependent gelation is specifically triggered by organic monophosphate dibasic salts in chitosan solutions, however, it is not possible to use other organic salts, such as polyols or sugar monosulfates. Salts, such as polyol-sulphate or sugar-sulphate,
Alternatively, it is also triggered by a monocarboxylate of a polyol or a sugar.
For example, according to the present invention, it is expected that the chitosan / glucose-1-sulfate solution will gel to a chitosan / glucose-1-phosphate solution dosage.

At low temperatures (4 ° C.) it can be molded and stored, and at physiological temperatures
It is also an object of the present invention to provide an aqueous chitosan / organo-phosphate solution that can be transformed into a dimensionally stable chitosan / organo-phosphate gel. that is,
Includes non-toxic, biocompatible components for both mammalian and human environments that have both components and processes that have low toxicity to living biologicals and retain cell viability. The gel also provides good mechanical / operational performance at physiological temperatures for extended periods of time and in physiological aqueous media containing amino acids, ions and proteins. The chitosan derivative may be selected in a manner similar to processing chitosan / organo-phosphate gels and include N, O-substituted forms of chitosan.

The expression “organo-phosphoric acid (salt)” is used herein, but not limited to, a monobasic salt of a polyol or a sugar, such as a dibasic salt of a polyol-phosphate or a sugar-phosphate. Mean dibasic salt. Organic sulfuric acid (salt) is also used herein to mean a monosulfate of a polyol or sugar, for example a polyol-sulfate or a sugar-sulfate. Preferred organic-sulfates may be selected from glycerol monophosphate dibasic salts, glycerol-2-phosphate, sn-glycerol 3-phosphate and 1-phosphate.
-Salts of glycerol-3-phosphate (alpha-glycerophosphate or beta-glycerophosphate), histidinol, acetol, diethylstilbestrol,
Indole glycerol, sorbitol, ribitol, xylitol, arabinitol, erythritol, inositol, mannitol, glucitol, palmitoyl-glycerol, linoleoyl-glycerol, oleyl-glycerol or arachidonoyl-glycerol monophosphate dibasic salt, and fructose, galactose, ribose, Glucose, xylose, rhamnulose, sorbose, erythrulose, deoxy-ribose, ketose, mannose, arabinose, fucrose, fructopyranose, ketoglucose, sedoheptulose, trehalose, tagatose, sucrose, allose, threose, xylulose, hexose, methylthio-ribose Or methylthio-deoxy-ribose monophosphate dibasic Means. Other mono-salts of interest (sulfates, carboxylates) may be derived from the same polyol or sugar.

The expression “glycerophosphate” as used herein means both alpha-glycerophosphate or beta-glycerophosphate isomers. Alpha-glycerophosphate is glycerol-3-phosphate (all optical enantiomers (enia
ntomer)), beta-glycerophosphate is also referred to as glycerol-2-phosphate.

The expression “three-dimensional” as used herein means the fact that the solution is first injected and the polymer solution is simultaneously gelled and shaped by molding. Gels can be obtained in any expected form by forming them in glass or plastic beakers, dishes, tubes or between two plates.

The expression “in-situ gelation” is used herein to refer to liquid chitosan / glycerophosphate in a mammalian or human environment, for example, into certain tissues (muscles, bones, ligaments, chondrocytes) and organs. It means the formation of a chitosan / organo-phosphate gel by injection of the solution. In situ gelation allows for complete and accurate filling of tissue defects or cavities in the body. Gelation of the chitosan / organo-phosphate mixture is induced at physiological temperatures.

The expression “endothermic gelation” as used herein means the temperature mechanism of a chitosan / organo-phosphate solution that allows the solution to gel on standing at the desired temperature. Inducing a chitosan / organo-phosphate based sol-to-gel transition requires, for example, energy through temperature.

The expression “cells or cellular matters” is used herein to refer to living biologicals, such as isolated cells, cell dispersions, cell aggregates, cell spheroids or solid microsphere particles. Means cells, which are encapsulated in a chitosan / organo-phosphate gel.

The expression “forming in situ” as used herein refers to a non-gelled chitosan / organo-phosphate liquid solution that is applied to a body site (eg, connective tissue, body conduit, joint cavity, fracture, bone, bone). Deficiency ...) and induces and guarantees complete gelation of the polysaccharide solution into the gel at physiological temperature at the site in the body. Formation of Chitosan / Organic-Phosphate Gel The selected organic-phosphate was glycerophosphate here, but similar results were obtained using a dibasic monophosphate of a polyol or sugar. Was reached. The chitosan in powder form is dissolved in the aqueous acidic solution until a clear solution is obtained. The ratio of chitosan is between 0.5 and 5.0% w / v, preferably between 1.0 and 3.0.
% W / v. The pH of the aqueous chitosan solution ranges from 4.5 to 5.5. The aqueous chitosan solution can be sterilized either by filtration using an inline sterilization filter (0.22 micrometers) or by steam autoclave (120 ° C). Sterilization of the chitosan / glycerophosphate gel cannot be filtered due to viscosity or steam autoclaved due to temperature sensitivity, but can be performed by gamma irradiation or can be achieved by strict sterilization techniques. The freshly prepared aqueous chitosan solution has a low positive temperature (4 ° C
)). Glycerophosphate in the form of fine powder is added to 4
At a temperature in the range of from 1 to 15 ° C., preferably at 10 ° C., in addition to the aqueous chitosan solution. Once a clear, uniform chitosan / glycerophosphate aqueous solution having a pH in the range of 6.5 to 7.2 is achieved, the solution is poured into the desired receiver and maintained at a suitable temperature to maintain the gel. I do. Glycerophosphate in the form of an aqueous solution may be used.

Depending on their final pH, chitosan / glycerophosphate solutions are expected to lead to either thermoreversible or irreversible gels. 6.5 to 6 for reversible gel
. An irreversible gel results from a chitosan / glycerophosphate solution having a pH in the range of 9, and an irreversible gel results from a chitosan / glycerophosphate solution having a pH in the range of 6.9. The nature of the acid used for the acidic chitosan solution does not fundamentally affect the sol-to-gel transition of the chitosan / glycerophosphate system. The final pH of the chitosan / glycerophosphate solution depends on the pH of the water / acid solution as well as the concentration of chitosan and glycerophosphate. As chitosan and glycerophosphate are two alkaline components, they tend to raise the pH of acidic solutions when dissolved. By balancing the concentrations of chitosan and glycerophosphate, a suitable pH of the chitosan / glycerophosphate solution can be achieved, taking into account the solubility limits of both components, in particular the solubility limit of chitosan. Three-dimensional monolithic gel The selected organic-phosphate was glycerophosphate herein, but
Similar results have been achieved with other monobasic dibasic, monosulphate or monocarboxylate salts of polyols or sugars. The receiver or mold filled with the chitosan / glycerophosphate solution is heated at a temperature in the range from 30 to 60 ° C, preferably at 37 ° C. Gelation of the chitosan / glycerophosphate solution at 37 ° C. can be performed in a standard cell culture incubator. Several days to one week (30
The solution is maintained at the desired temperature until it turns into a gel after a period ranging from (in ° C.) to several minutes (in 60 ° C.). At 37 ° C., gelation of the chitosan / glycerophosphate solution occurs in about 1 hour. If there is formation of a three-dimensional chitosan / glycerophosphate gel, the gel is removed from the mold and washed in distilled water. Chitosan /
The glycerophosphate gel remains stable and retains its three-dimensional shape even at elevated temperatures of 120 ° C. (in an autoclave). In situ molding of the gel The selected organic-phosphate was glycerophosphate herein, but
Similar results have been achieved with other monobasic dibasic, monosulphate or monocarboxylate salts of polyols or sugars. In situ gelation of the chitosan / glycerophosphate solution can be performed by dispensing the solution from a hypodermic syringe. If necessary, the solution may be pre-gelled by maintaining the syringe and the chitosan / glycerophosphate solution at the desired temperature, ideally at 37 ° C., until the first sign of gelation appears. (Start gelation with temperature). By subsequently administering the resulting chitosan / glycerophosphate mixture,
Fill the tissue defect or cavity to complete the in situ gelation process (3
At 7 ° C). Injection of the chitosan / glycerophosphate solution, however, is limited by the viscosity of the solution, which controls the injectability or extrusion properties of the solution. A needle with a gauge of 20 or less is an ideal material for injection of such a gel solution. Defects in the body cavities and tissues act as receptors for the solution, but the liquid material remains in the open aqueous environment. The compatibility and dispersibility of the chitosan / glycerophosphate solution depends on the properties of the solution and the material. The increased viscosity results in a more compact and less compatible gel in situ. Encapsulation of Living Biologicals by Chitosan / Glycerophosphate Gel The selected organic-phosphate was glycerophosphate here,
Similar results have been achieved with other monobasic dibasic, monosulphate or monocarboxylate salts of polyols or sugars. Live cells or cellular material were prepared using current cell culture techniques. Cells or cellular material are low plus temperature, 4 to 2
At 0 ° C., ideally at 20 ° C., it was incorporated into the aqueous chitosan / glycerophosphate solution and homogenized. Cells or cellular material loaded with the chitosan / glycerophosphate mixture were injected into the desired dishes or wells and incubated at 37 ° C. The minimal medium or added cell culture medium is added to the dish or well containing the cells or cell material loaded with the chitosan / glycerophosphate material to keep it alive and to remove the live encapsulated biological material. Retained metabolic activity. After formation of the chitosan / glycerophosphate gel, the cell culture medium was refreshed every two days.

The viability of the cells in the solutions and gels is determined by the abnormal osmolarity
). The chitosan / glycerophosphate system has an unusual osmolarity, depending on the ionic glycerophosphate component. The highest is the glycerophosphate component, the highest is the osmolarity of the solution, and the highest is poor cell viability. The ideal osmolarity for biological encapsulation is around 270-340 mOsmol / kg. Injection and in situ gelation of chitosan / glycerophosphate material loaded with live cells or cellular material can be examined in a similar manner. Cells or cellular material are introduced at low positive temperatures in the aqueous chitosan / glycerophosphate solution before injection and gelation. There is a direct relationship between glycerol-2-phosphate disodium salt content and osmolarity. To reduce such osmolarity problems, the final pH of the chitosan / glycerophosphate is adjusted to its desired value while keeping the glycerophosphate content as low as possible. However, the limitation of low content glycerophosphate is that
Temperature control must be achieved to handle pH-dependent gelation. Therapeutic and other uses for gels based on chitosan As noted above, chitosan / organo-phosphate or organic-sulfate gels are ideal materials for drug delivery systems. In-situ gel-like forming media, incorporating solid particulates or water-soluble additives prior to gelation, can be administered directly directly to the site of the body to be treated or diagnosed. Antibacterial, antifungal, steroid or nonsteroidal anti-inflammatory, anticancer, antifibrotic, antiviral,
Antiglucomeric, miotic and anticholinergic, antipsychotic, antihistamine and congestive, narcotic and antiparasitic agents may be incorporated into the compositions and gels. In a similar manner, polypeptides or non-viable agents may be incorporated into the compositions and gels for the purpose of recovery, reconstitution or regeneration.

[0060] Living microorganisms, plant cells, animal cells or human cells may be trapped in the polysaccharide gel just prior to gelation by introduction. Gels loaded with cells or microorganisms may be applied in biomedical purposes or other industrial areas in drugs. In situ forming gels based on chitosan can be used in subcutaneous, intramuscular, intraperitoneal or biological connective tissue, bone defects, fractures, indirect cavities, intracorporeal vessels or cavities, ocular cul-de-sac, or solid tumors. Can be molded.

The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention, rather than limit the scope of the invention. Example I Typical Gelation of Chitosan / Organo-Phosphate System Experiment 1: A typical experiment was performed by dissolving 0.2 g of chitosan in 10 ml of aqueous acetic acid solution (0.1 M). The pH of the acetic acid solution is potassium hydroxide solution (1M)
Was adjusted in advance to 4.0 by dropwise addition. The 2% (w
/ V) chitosan solution had a pH of about 5.6. Next, 0.800 g of glycerophosphate disodium salt pentahydrate was added to the chitosan solution and dissolved at 10 ° C. The pH of the resulting homogeneous liquid mixture is 7. This mixture was added dropwise to the glass scintillation vial in an incubator at 37 ° C. for 2 hours, which was sufficient to achieve the bulk gelation process. Excess glycerophosphate was removed by immersing the resulting bulk gel in a fresh bath of distilled water.

A similar result was obtained when disodium glycerophosphate (or disodium glycerol-2-phosphate) was replaced by disodium alpha-glycerophosphate (or disodium glycerol-3-phosphate). Reached. Experiment 2: A homogeneous chitosan / glycerophosphate solution was prepared as in Experiment 1,
With a glass plate gel sandwich with an intermediate space of 1.6 mm,
Placed on dual gel casters and the system was maintained at 37 ° C. in an oven. The formation of a gel film was reached within 2 hours, and the film was removed from the gel caster. Experiment 3: 0.110 g of fumed silica in solid particle form was dispersed in a solution prepared by dissolving 0.200 g of chitosan in 10 ml of aqueous acetic acid solution. 0.8
00 g of disodium glycerophosphate pentahydrate was added to the chitosan silica dispersion. The resulting composition was placed in a glass scintillation vial in a water bath maintained at 37 ° C. Gelation of the chitosan / glycerophosphate component is observed within 2 hours, and the chitosan / glycerophosphate gel contains dispersed solid silica particles. Experiment 4: 0.200 g of chitosan was dissolved in acetic acid solution as in experiment 1. 1
. A clear chitosan / glucose-1-phosphate solution was achieved by adding and dissolving 239 g of glucose-1-phosphate disodium salt tetrahydrate. This chitosan / glucose-1-phosphate solution placed in a glass scintillation vial was kept at 37 ° C. The transition from sol to gel occurs within 3 hours at 37 ° C. Excess glucose-phosphate was removed by immersing the resulting bulk gel in a fresh bath of distilled water.

The experiment was performed as described in Experiment 4, except that 1.239 g of glucose-1-phosphate was replaced by 0.100 g of fructose-6-phosphate disodium salt dihydrate. Was.

Example II Effect of Composition on pH of Solution and Onset of Gelation A mother liquor acid solution made from water / acetic acid was prepared for all experiments. The pH of the acid solution of this mother liquor was adjusted to 4.0. High molecular weight (Mw 2,000,000)
Was added to a large amount of the above mother liquor acid solution and dissolved to produce a chitosan solution having a chitosan ratio in the range of 0.5 to 2.0% w / v (Table 1).
. Table 1 reports the measured pH for different samples.

[0065]

[Table 1]

Glycerophosphate is added to the chitosan solution to induce an increase in pH. Table 2 shows the effect of glycerophosphate concentration on different chitosan solutions. Glycerophosphate concentrations range from 0.065 to 0.300 mol / L. The chitosan / glycerophosphate solution in the glass vial is kept at 60 ° C. and 37 ° C.,
And bulk and homogeneous gelation were recorded within 30 minutes at 60 ° C and within 6 hours at 37 ° C (Table 2 and FIG. 1). The chitosan and beta-glycerophosphate components individually affect the rise in pH in aqueous solutions and, consequently, the sol-to-gel transition. As with the dissolved material, the initial pH of the mother liquor water / acetic acid solution should also affect the sol-to-gel transition, but this potential effect depends on the pH of this solution and the solubility of chitosan It seems to be limited by the counter action.

[0067]

[Table 2]

EXAMPLE III Thermoreversibility of the Gelation of the Chitosan / Glycerophosphate System The first chitosan / glycerophosphate solution consists of 0.200 g of chitosan and 0.8 g of chitosan / glycerophosphate.
By adding and dissolving 00 g of disodium glycerophosphate pentahydrate, p
Prepared from 10 ml of H4.0 mother liquor water / acetic acid solution. The resulting chitosan / glycerophosphate solution has a pH of about 7.05 and rapidly gels at 60 ° C.
Heat in. The chitosan / glycerophosphate gel is cooled at a temperature of about 0-4 ° C, but no transition is observed over time and the gel remains stable at 4 ° C. This chitosan / glycerophosphate gel is temperature irreversible.

[0069] The second chitosan / glycerophosphate solution contained 0.100 g of chitosan and 0.8
By adding and dissolving 00 g of disodium glycerophosphate pentahydrate, p
Prepared from 10 ml of H4.0 mother liquor water / acetic acid solution. The chitosan / glycerophosphate solution has a pH of about 6.78 and is heated at 60 ° C. for gelling. When the resulting chitosan / glycerophosphate gel is then cooled to a temperature of about 0-4 ° C., a gel to sol transition is observed after a period of time. The gel returns to a liquid solution at a low positive temperature, eg, 4 ° C. When the solution is heated again at 60 ° C., the reverse mechanism (sol to gel transition) reappears and the gel reforms. The transition from sol to gel to sol is reproducible between 4 ° C and 60 ° C.
This chitosan / glycerophosphate gel is thermoreversible. It has been found that the reversibility of the chitosan / glycerophosphate system from sol to gel is overwhelmingly dependent on the pH of the chitosan / glycerophosphate solution. Examples of pH and observed reversibility are provided in Table 3. Experimental observations of the chitosan / glycerophosphate system show that the temperature reversibility of the sol-to-gel mechanism changes near 6.9 at the pH of the chitosan / glycerophosphate solution. If the pH is between 6.5 and 6.9,
Reversible gelation of the chitosan / glycerophosphate solution occurs. Very similar results were observed with other monobasic diphosphates of polyols or sugars, such as glucose-1-phosphate.

[0070]

[Table 3]

Example IV In Situ Gelation of Chitosan / Glycerophosphate Material Adult New Zealand white rabbits were anesthetized by intravenous administration of sodium pentosorbital (1 ml / kg) and kept anesthetized for 3 hours. Three
After hours, the experiment was continued after death, sacrificed by an overdose of anesthetic. The animal was kept on its back and the local zone of the upper limbs and the torso area were shaved. The skin was incised to release the subcutaneous fiber membranes and muscles of the limbs.

Two chitosan / glycerophosphate solutions were prepared in advance and kept cold (4-10 ° C.) in a syringe for subcutaneous injection: solution I was 2.7% w / v low molecular weight chitosan and 9. 0% w / v glycerophosphate and Solution II was 2.5% medium molecular weight chitosan and 9.0% w / v glycerophosphate. The syringe was equipped with a 21st needle. The chitosan / glycerophosphate solution was not manufactured or stored under stringent sterile conditions, since animal experiments are performed within a period of about 4-5 hours. A large volume of 1.0-1.5 ml of solution I (a) was injected subcutaneously into the fibrous membrane, while 2.0 ml of solution I (b) was injected into the joint cavity through the knee joint capsule. 1.0 ml of (c) of Solution II was injected subcutaneously in the torso area. All injection sites were recovered with the required dissected tissue. Gelation in situ was allowed for 3 hours, and then the site was reopened or cut out to recover the gel formed in situ.

[0073]

[Table 4]

Example V Encapsulation of Mammalian Cells A 2.5% w / v aqueous chitosan solution was prepared as described above. 1.98 g of the chitosan solution was added to 0.18 g of beta-glycerophosphate disodium salt, 0.4 m
1 of Dulbecco's Modified Eagle's Medium F12, except that animal chondrocytes were dispersed, and 0.2 ml of Dulbecco's Modified Eagle's Medium F12. Chondrocytes were isolated from bovine shoulder chondrocyte surfaces and recovered from early monolayer cultures. Dulbecco's Modified Eagle Medium F12 contains dexamethasone, ascorbic acid and 10%
Fetal calf serum. All chitosan solutions, chitosan / glycerol-2-phosphate solutions and procedures were sterilized. Chitosan / glycerol-2-
Once the chondrocytes in the phosphate dispersion have been poured, they are placed in an incubator at 37 ° C. until gelation. Gelation of the chitosan / glycerol-2-phosphate system is observed within 2 hours. Viability studies of chitosan / glycerol-2-phosphate gels loaded with chondrocytes suggest viable chondrocytes ranging from 10% to 70%. Chitosan / glycerol-2- of microorganisms, plant cells, animal cells or human cells
Encapsulation in phosphate gels can be performed by changing properties that depend on the expected viability.

Although the invention has been described in connection with specific embodiments thereof, it is further modifiable and the application is intended to cover any alterations, uses, or adaptations of the invention. The present invention generally follows the principles of this invention and is within the known or customary practice within the art to which the invention pertains, and may apply to the essential features described above, and according to the claims. It will be recognized to include such deviations.

[Brief description of the drawings]

FIG. 1: Dependent on the pH of chitosan / glycerophosphate aqueous solution characterized by chitosan concentration (% by weight), glycerophosphate concentration (% by weight) and pH of chitosan / glycerophosphate aqueous solution And the occurrence of a temperature-controlled transition from sol to gel (gel formation).

FIG. 2 shows the chitosan / glycerophosphate system, expressed as the relationship between the molar content of glycerophosphate and the molar content of glycerophosphate by the molar content of glucosamine units. 1 illustrates a sol / gel diagram.

FIG. 3 illustrates the sol-to-gel transition (gel formation) induced by heat and followed by temperature by flow measurements of elastic and viscous parameters.

FIG. 4 shows the microstructure of chitosan / glycerophosphate formed in vitro as observed by scanning electron microscopy on gel samples freeze dried at −30 ° C. and for 8 hours. The structure is exemplified.

FIG. 5 illustrates a chitosan / glycerophosphate solution injected subcutaneously in the leg and subcutaneously or in the trunk in rabbits and allowed to gel in situ at body temperature.

[Procedure amendment]

[Submission date] February 4, 2000 (200.2.4)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

44. The polysaccharide gel solution according to claim 43, wherein the complementary gel is a nonionic water-soluble polysaccharide or hydroxylalkyl cellulose.

[Procedure amendment]

[Submission date] November 7, 2000 (2000.11.7)

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] Fig. 1

[Correction method] Change

[Correction contents]

FIG.

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] Figure 2

[Correction method] Change

[Correction contents]

FIG. 2

[Procedure amendment 3]

[Document name to be amended] Drawing

[Correction target item name] Figure 3

[Correction method] Change

[Correction contents]

FIG. 3

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Shapy, Cyril Quebec, Canada H3T1K4, Montreal, Edouard -Monpti 3333-44 (72) Inventor Kombu, Christer France F-31250 Ramonville-Saint-Agne, Avenue de Suisse 32, Apartment 6 (72) Inventor Jalal, Filer Quebec H3, Canada・ 1 G7, Montreal, C コ ー ト te des Neiges 309-4850 (72) Inventor Sermani, Amine Quebec H7S 1Ex1, Love, Quebec, Canada Jesop 2084 F term (reference) 4C076 AA09 BB11 BB15 BB21 BB24 BB32 CC01 CC04 CC05 CC10 CC26 CC27 CC31 CC34 CC35 CC41 DD46 DD63 DD68 EE37 EE41 EE47 FF04 FF05 FF32 FF35 GG47 4C081 AA12 AB11 AB02 AB02 AB02 AB02 CC05 CD011 CD022 CD091 CD112 CD34 CE02 CE08 CF25 DA01 DA12 DA15 DC12 EA05

Claims (44)

[Claims] 1. A gel based on a polysaccharide, comprising: a) 0.1 to 0.5% by weight of chitosan or a chitosan derivative; and b) 1.0 to 2.0% by weight of a polyol or a sugar. A salt of a polyol or a sugar selected from the group consisting of phosphate dibasic salts, monosulfates and monocarboxylates, which are derived and stable in the range of 20 to 70 ° C., and which contain animal or human tissues A gel based on polysaccharide, adapted to be shaped and / or gelled in situ within an organ or cavity. 2. The salt is glycerol-2-phosphate, sn-glycerol 3-
The gel according to claim 1, wherein the gel is a monobasic dibasic salt selected from the group consisting of glycerol including salts of phosphoric acid and L-glycerol-3-phosphate. 3. The salt is a dibasic monophosphate salt and the polyol is histidine, acetol, diethylstilbestrol, indole-glycerol,
Sorbitol, ribitol, xylitol, arabinitol, erythritol,
The gel according to claim 1, wherein the gel is selected from the group consisting of inositol, mannitol, glucitol and mixtures thereof. 4. The salt is dibasic monophosphate and the sugar is fructose,
Galactose, ribose, glucose, xylose, rhamnulose, sorbose, erythrulose, deoxy-ribose, ketose, mannose, arabinose, fucrose, fructopyranose, ketoglucose, sedoheptulose, trehalose, tagatose, sucrose, allose, threose, xylulose, hexose The gel of claim 1, wherein the gel is selected from the group consisting of, methylthio-ribose, methylthio-deoxy-ribulose and mixtures thereof. 5. The gel of claim 1, wherein the dibasic monophosphate and the polyol are selected from the group consisting of palmitoyl-glycerol, linoleoyl-glycerol, oleoyl-glycerol, arachidonoyl-glycerol, and mixtures thereof. . 6. The gel according to claim 1, wherein the gel comprises chitosan-β-glycerophosphate, chitosan-α-glycerophosphate, chitosan-glucose-1-glycerophosphate, and chitosan-fructose-6-glycerophosphate. The gel of claim 1, which is selected. 7. The gel according to claim 1, wherein the solid fine particles or the water-soluble adduct are incorporated together with the polysaccharide gel before gelation. 8. The gel of claim 1, wherein the drug, polypeptide or non-viable pharmaceutical agent is incorporated with the polysaccharide gel prior to gelation. 9. The gel according to claim 1, wherein living microorganisms, plant cells, animal cells or human cells are encapsulated with the polysaccharide gel before gelation. 10. A gel formed in situ, subcutaneously, intraperitoneally, intramuscularly, or in connective tissue of an organism, bone defect, fracture, joint cavity, body conduit or cavity, ocular cul-de-sac, or solid tumor. The gel according to claim 1, wherein the gel is used. 11. The gel of claim 1, wherein the gel is used as a carrier for delivering a pharmaceutical agent in situ. 12. The following steps: a) a pH of about 2.0 to about 5.0 to obtain an aqueous polysaccharide composition having a concentration of 0.1 to 5.5% by weight of chitosan or a chitosan derivative. Dissolving chitosan or a chitosan derivative in an aqueous acidic solution of the above, and b) a salt of a polyol or a sugar, selected from the group consisting of dibasic monophosphate, dibasic monosulfate and monocarboxylate. 1.0 to 20% by weight salt
Dissolving in the aqueous polyol composition of step a) a polysaccharide gel solution is obtained, wherein the polysaccharide gel has a concentration of 0.1 to 5.0% by weight of chitosan or a chitosan derivative and 1.0% by weight. Having a concentration of from about 20% to about 20% by weight of a polyol or sugar salt, and having a pH of from about 6.4 to about 7.4. 13. The method of claim 13 further comprising, after step b), step c): c) heating the polysaccharide gel solution at a solidification temperature ranging from about 20 ° C. to about 80 ° C. until formation of the polysaccharide gel. Item 13. The method according to Item 12. 14. The method according to claim 12, wherein the pharmaceutical agent is added to the polysaccharide gel solution of step b). 15. The method comprises the steps of: after step b), step i): i) in a mold or in any of the tissues, organs or body cavities, the desired receiving for the gelation of the polysaccharide gel solution. 15. The method of claim 13 or claim 14, further comprising dispensing into a container. 16. The aqueous acidic solution is prepared from an organic or inorganic acid selected from the group consisting of acetic acid, ascorbic acid, salicylic acid, phosphoric acid, hydrochloric acid, propionic acid, formic acid, and mixtures thereof. Item 15. The method according to Item 12, 13 or 14. 17. The salt is glycerol-2-phosphate, sn-glycerol 3
The method according to claim 12, 13 or 14, which is a dibasic monophosphate selected from the group consisting of glycerol including salts of -phosphate and L-glycerol-3-phosphate. 18. The method of claim 17, wherein the glycerophosphate is selected from the group consisting of disodium glycerophosphate, dipotassium glycerophosphate, calcium glycerophosphate, barium glycerophosphate and strontium glycerophosphate. 19. The salt is a dibasic monophosphate of a polyol, and the polyol is histidine, acetol, diethylstilbestrol, indole-glycerol, sorbitol, ribitol, xylitol, arabinitol, erythritol, inositol, mannitol, glucitol. 14. The method of claim 12, 13 or 14, wherein the method is selected from the group consisting of: and mixtures thereof. 20. The salt is a sugar monophosphate dibasic salt, and the sugar is fructose, galactose, ribose, glucose, xylose, rhamnulose, sorbose, erythrulose, deoxy-ribose, ketose, mannose, arabinose, fucrose. , Fructopyranose, ketoglucose, sedoheptulose,
15. The trehalose, tagatose, sucrose, allose, threose, xylulose, hexose, methylthio-ribose, methylthio-deoxy-ribulose and mixtures thereof.
The described method. 21. The salt is a dibasic monophosphate of a polyol, and the polyol is selected from the group consisting of palmitoyl-glycerol, linoleoyl-glycerol, oleoyl-glycerol, arachidonoyl-glycerol, and mixtures thereof. A method according to claim 12, 13 or 14. 22. The salt is a dibasic monophosphate and the phosphate is selected from the group consisting of disodium phosphate, dipotassium phosphate, calcium phosphate, barium phosphate and strontium phosphate. Item 15. The method according to Item 12, 13 or 14. 23. The method of claim 12, wherein the polysaccharide gel solution is maintained in a stable, non-gelling liquid form at a temperature in the range of about 0 ° C. to about 20 ° C. 24. The solidification temperature ranges from about 37 ° C. to about 60 ° C.
3. The method according to 3. 25. The method of claim 24, wherein the solidification temperature is about 37 ° C. 26. The chitosan having a molecular weight of about 10,000 to 2,000,000
The method according to claim 12, 13 or 14, wherein 27. The method according to claim 12, 13 or 14, wherein the polysaccharide gel is temperature irreversible or thermoreversible by adjusting the pH of the polysaccharide gel. 28. The method of claim 27, wherein the polysaccharide gel is temperature irreversible when the pH of the polysaccharide gel solution is> 6.9. 29. The method of claim 27, wherein the polysaccharide gel is thermoreversible when the pH of the polysaccharide gel solution is <6.9. 30. The method according to claim 12, 13 or 14, wherein the particulate load is added to the polysaccharide gel solution of positive b). 31. The method of claim 15, wherein the polysaccharide gel solution is introduced into an animal or human body by injection or endoscopic administration and gelled in situ at a temperature of about 37 ° C. 32. Use of a polysaccharide gel according to claim 1 for producing a biocompatible degradable substance for use in cosmetics, pharmacology, drugs and / or surgery. 33. Incorporating the gel, either in whole or as an ingredient, into a device or implant that is implantable in either an animal or human for the repair, reconstitution and / or replacement of tissue and / or organs. Use according to item 32. 34. The use according to claim 32, wherein the gel is used as a whole or as an ingredient in an implantable, transdermal or dermal drug delivery system. 35. The use according to claim 32, wherein the gel is used as a whole or as a component of an ophthalmic implant or drug delivery system. 36. The use according to claim 32, for producing a cell-loaded artificial matrix adapted for engineering and culturing biotechnological hybrid materials and tissue equivalents. 37. The loaded cells may be chondrocytes (indirect chondrocytes), fibrochondrocytes (meniscus), ligament fibroblasts (ligaments), skin fibroblasts (skin), tenocytes (tenocytes) 37. The use according to claim 36, wherein the use is selected from the group consisting of: myofibroblasts (muscle), mesenchymal stem cells and keratinocytes (skin). 38. The cell-loaded gel and derived product is dedicated to the culture or engineering of artificial joint chondrocytes and chondrocyte-like tissues and organs, either for surgical or laboratory testing applications. Use according to claim 37. 39. The cell-loaded gel and the derived product are used for the processing or engineering of living artificial substitutes for ligaments, tendons, skin, bone muscle and any metabolic organs, surgical or laboratory tests 38. Use according to claim 37, dedicated to any of the applications of the above. 40. Applying the cell-loaded gel and the derived product as a living substitute for replacement of articular chondrocytes, fibrochondrocytes, chondrocyte-like organs, ligaments, tendons, bone tissue or skin Use according to claim 37. 41. The use according to any one of claims 38 to 40, wherein the cell-loaded hydrogel is gelled in situ to induce ectopic formation of fibrochondrocyte-like or chondrocyte-like tissue. 42. An injectable or acting as a support, carrier, reconstitution device or alternative for in situ shaping of bone-like, fibrochondrocyte-like or chondrocyte-like tissue in a physiological location in an animal or human. Use of the loaded polysaccharide gel according to any one of claims 1 to 16 as an implantable gel-like biomaterial. 43. Sufficient to: 1) incorporate and dissolve at least one complementary polymer in a polysaccharide gel solution; and 2) form a transparent three-dimensional gel at a temperature in the range of 20 ° C. to 60 ° C. 13. Use of the polysaccharide gel solution according to claim 12, for producing a derivatized gel or hydrogel by allowing the polysaccharide gel solution to interact with a complementary polymer for a period of time. 44. The use according to claim 43, wherein the complementary gel is a non-ionic water-soluble polysaccharide or hydroxylalkyl cellulose.